Immunoassays, which take advantage of natural immunological reactions, have found widespread use as analytical techniques in clinical chemistry. Because of the specificity of the reactions, they are particularly advantageous in quantifying biological analytes (called ligands herein) which include, for example, antibodies therapeutic drugs, narcotics, enzymes, hormones, proteins, etc.
In competitive binding assays, a labeled ligand analog (sometimes referred to as ligand analog herein) is placed in competition with the unlabeled ligand for reaction with a fixed amount of the appropriate binding material (called a receptor herein). Unknown concentrations of the ligand can be determined from the measured signal of either the bound or unbound (i.e. free) ligand analog. The reaction proceeds as follows:
ligand+ligand analog+receptor .revreaction.ligand-receptor+ligand-analog receptor. PA1 (a) providing a mono- or a polysaccharide having a group capable of specific binding with an immunologically reactive analyte (ligand) and two vicinal diols located on a terminal saccharide group; and PA1 (b) oxidizing said terminal saccharide at the vicinal diol portion with a mixture of periodic acid and chromium trioxide in aqueous mixtures of miscible organic solvents to produce the oxidation product.
The traditional approach to the preparation of labeled ligand analogs for haptens such as steroids which contain carbohydrate residues with vicinal diols on the terminal monosaccharide residues, involves the oxidation of the terminal monosaccharide residues of steroids to dialdehydes followed by attachment to amine residues of an enzyme label. The problem is that this approach has not generated satisfactory enzyme labeled ligand analogs for immunoassays. In such assays a substantial amount of the analog is not bound by antibody. Attempts to functionalize digoxigenin, the steroid component of digoxin, have met with limited success due to limited recognition of such functionalized material by the antibody. It would be desirable to have functionalized steroid derivatives such as digoxin that are easily bound to labels such as enzymes and that are recognized by antibodies for the steroid.